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November 2014

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A-/- mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitroand in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin.

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VCU Microbiology and Immunology Publications

Additional Files

Figure_S1.tif (1469 kB) SP-A-degrading ability is reduced in ΔlasB mutant bacteria in vitro. (A) hSP-A (25 µg) was incubated with 1×108 PAO1, ΔlasB or PDO240LasB bacteria in LB supplemented with 0.6 mM ZnCl2 for the indicated time intervals. hSP-A degradation was assessed by western blot analyses using 10 µl of SP-A/bacterial suspension. Image from one of the three independent experiments is shown. (B) hSP-A degradation P. aeruginosa strains in the absence of ZnCl2. Immunoblots were probed with anti-SP-A antibody.